PCR typing of tetracycline resistance determinants (Tet A–E) in Salmonella enterica serotype Hadar and in the microbial community of activated sludges from hospital and urban wastewater treatment facilities in Belgium

Abstract

The distribution of tetracycline resistance determinants Tet A^E was studied by PCR in 40 tetracycline-resistant Salmonella enterica serotype Hadar (S. hadar) isolates collected from human patients in 1996 and 1997, as well as in the microbial community originating from activated sludges of hospital and urban wastewater treatment facilities. A fast DNA extraction and purification method from activated sludges was used to provide amplifiable DNA. The method is based on the direct lysis of bacteria improved by bead-beating followed by DNA purification on polyvinylpolypyrrolidone spin columns to remove PCR inhibitors. The purified DNAs from salmonellae and activated sludges were characterized for the presence of tetracycline determinants with specific primer pairs designed on the basis of published sequences. The Tet A determinant was present in all clinical isolates and DNAs extracted from the bacterial community of the selected activated sludges. The Tet C determinant was identified in only one of the 40 clinical isolates and in six of the seven environmental samples. No signal was detected for Tet B, D and E determinants. This study revealed a high and stable prevalence of the Tet A determinant in both salmonellae clinical isolates and the microbial community of activated sludges from hospital and urban wastewater treatment facilities over a 2-year period. ß

Similarity of Tetracycline Resistance Genes Isolated from Fish Farm Bacteria to Those from Clinical Isolates

“…These bacteria accounted for about 4.5% of all Tet r strains. This value was higher than those previously reported for lactose-fermenting coliforms (27), Salmonella serovar Typhimurium (28), Salmonella serovar Hadar (16), Aeromonas, and Enterobacteriaceae isolated from catfish, catfish ponds, and rainbow trout farms (12,13,14,37). Although environmental factors have been reported to affect the number of resistance genes carried by a single cell (11), the difference is more likely to be caused by a difference in the number of PCR primers utilized.…”

“…Dissemination of the proton-dependent tetracycline efflux protein in aquaculture environments has been reported (5,12,13,14,22,34,37). Previous work has identified the relevant genes by using DNA hybridization or PCR methods (4,12,13,14,15,16,27,28,34,37), but the nucleotide sequences of these determinants remain unknown. In the present study, we isolated numerous Tet r gram-negative fish farm bacteria and determined the DNA sequences of the Tet r genes.…”

Genes encoding tetracycline resistance in a full-scale municipal wastewater treatment plant investigated during one year

“…In wastewater treatment plants (WWTPs) tetracyclines have been detected both in the influent and effluent (Karthikeyan & Meyer 2006). Bacteria resistant to tetracycline and genes encoding tetracycline resistance (tet-genes) are also commonly described in WWTPs (Guillaume et al 2000;Auerbach et al 2007;Zwenger & Gillock 2009). One study has shown that the fraction of tetracycline-resistant bacteria increased with increasing concentrations of influent tetracycline (Kim et al 2007a,b).…”

Development and Application of Real-Time PCR Assays for Quantification of Genes Encoding Tetracycline Resistance

“…The application of PCR to detecting AR genes in both bacterial isolates and environmental samples has provided additional insights into the occurrence of AR in various environments (4,5,9,15,24). The demonstration that AR genes are detectable in groundwater samples collected downstream of livestock production environments and animal waste lagoons (4,9) has further heightened concerns about the role of agriculture in the dissemination of AR (13,32).…”